The Ail family of proteins has been shown to mediate cell adhesion and resistance to human serum in several pathogenic Yersinia species. We have recently shown that Ail is important for delivery of cytotoxic Yop proteins from Yersinia pestis to phagocytic and non-phagocytic human cells. This defect in Yop delivery in vitro is reflected in the >3,000-fold increase in LD50 of a (ail mutant of Y. pestis KIM5 by the intravenous route of infection. Along with the decreased virulence of the (ail mutant, we also observed increased inflammation within infected spleen and liver tissues based on histology and greatly decreased bacterial loads in these tissues three days post- infection. This is the expected result if Yop delivery is inhibited, leading to lack of delivery of anti-inflammatory Yops. Due to the major role of Ail in plague pathogenesis, we propose to use a recently purified Ail protein to determine the ability of Ail immunization to protect naive mice from wild-type (fully virulent) Y. pestis infection delivered subcutaneously (bubonic plague) or intranasally (pneumonic plague). Given that experimental immunization with V antigen of Y. pestis or the non-immunosuppressive derivative V10 are standards in the field for plague vaccination, we will also characterize the combined protection provided by Ail together with V10. Y. pestis is the etiological agent of plague, a rapidly fatal disease and potential bioterrorism threat. There is currently no licensed plague vaccine in the U.S. and recent reports indicate escape mutants may undermine the efficacy of vaccines utilizing the F1 capsule as an immunogen. Thus, identification and development of additional protective antigens for future plague vaccine formulations are essential. Given the critical role for Y. pestis Ail in Yop delivery and virulence, it is an excellent candidate antigen for immunization trials using an animal model. The specific Aims of this proposal are: 1. Assess the efficacy of Ail immunization for preventing plague in mice 2. Assess the efficacy of Ail + V10 (a derivative of V antigen) for cumulative plague protection in mice PUBLIC HEALTH RELEVANCE: This project will test the efficacy of a new vaccine antigen, Ail, for its ability to protect mice from plague. If Ail proves to protect animals from disease, it could be incorporated into future vaccine formulations to prevent plague in humans. Plague is a rapidly-fatal infectious disease and potential bioterrorism threat with no currently licensed vaccine in the U.S.